The goal of this research program is to exploit, and further develop, techniques for manipulating neural activity to identify the brain circuits underlying specific behaviors. Using the Gal4-UAS gene targeting system of Drosophila melanogaster to drive the expression of genes whose products inhibit neuronal excitability, we are selectively suppressing the activity of subsets of neurons and analyzing the effects of this manipulation on behavior. We are particularly interested in the suite of hormonally coordinated and developmentally programmed behaviors executed by the adult fly shortly after emergence from the pupal case, with an immediate focus on those necessary for wing expansion. To identify neuronal substrates of these behaviors we use two approaches: one in which defined subsets of neurons, and the other in which random subsets, are inhibited (using Gal4 lines with defined promoters and Gal4 enhancer-trap lines, respectively). In each case, patterns of suppression that affect the behaviors of interest are identified for further characterization. Using the first approach, we have found that inhibition of electrical activity in a small subset of neurons that express the neuromodulator CCAP suppresses wing expansion in flies. Using the second approach, we have identified 27 patterns of suppression (i.e. enhancer-trap lines) that generate wing expansion deficits. Using a transgenic line that specifically suppresses Gal4 activity in CCAP-expressing neurons (CCAP-Gal80), we have demonstrated that 26 of these enhancer-trap lines exert their effects on wing expansion by acting within the CCAP neurons. One of these lines expresses primarily in a subset of CCAP-expressing neurons that also expresses the hormone bursicon, which is known from genetic studies to be required for wing expansion. We used this line to demonstrate that there are two functionally distinct groups of CCAP-expressing neurons. One group (the output group) is responsible for secreting bursicon into the hemolymph (blood); the other group (the regulatory group) modulates the activity of the output group. Having established that the CCAP-expressing neurons form a network that controls bursicon release, our goal is to provide a complete functional characterization of this network. On-going work with other enhancer-trap lines is designed to establish the functional identities of further subsets of CCAP-expressing neurons. To facilitate investigation of the CCAP network, we have developed two techniques that should be generally applicable to the targeted manipulation of neuronal function. The first technique permits the targeted enhancement of excitability using the gene encoding the bacterial sodium channel, NaChBac, fused to Green Fluorescent Protein (GFP). We have shown that NaChBac-GFP enhances excitability in Drosophila muscles and neurons and have used it to demonstrate that enhanced excitability in the regulatory group of CCAP-expressing neurons disrupts bursicon secretion. The second tool we have developed is a modification of the Gal4-UAS technique that incorporates technology from the yeast two-hybrid system. We have split the Gal4 molecule into its component DNA-binding (DB) and transcription activation (TA) domains and fused them to heterodimerizing leucine zippers. This permits the DB and TA domains to associate in cells that express both domains and reconstitute Gal4 transcriptional activity. Cells expressing a single domain lack this activity. By independently targeting the two domains in vivo, we can activate UAS transgenes selectively in the subset of cells that expresses both domains. This has allowed us to rationally restrict our manipulations of neural function to specific subsets of the CCAP network. We anticipate that this tool will find broad use in the refined targeting of genetic manipulations to small subsets of cells. Investigation of the neuronal substrates of posteclosion behavior in Drosophila using the broad palette of tools we are developing should serve as a ?proof of concept? of a circuit mapping approach that can later be extended to studies of mammalian behavior.